Cancer is a disease state characterized by the uncontrolled proliferation of genetically altered tissue cells. There have been several chemotherapeutic approaches developed to target cancer. These include alkylating and anti-mitotic agents, anti-metabolites and anti-tumor antibiotics. Such therapeutic agents act preferentially on rapidly proliferating cells such as cancer cells.
Acquired resistance mediated by DNA repair enzymes has often imposed severe limitations on the use of DNA-interactive agents and, in many cases, useful clinical anti-tumor activity could not be observed with the administration of multiple anti-tumor drugs having different mechanisms of action.
In the last 10 years there has been a considerable increase in our understanding of the molecular basis of cellular resistance via DNA repair processes, as well as of the spontaneous repair of naturally occurring errors in DNA polymerization. However, to date this has not translated into an increase in the efficacy of DNA-reactive drugs targeting cancers, including breast, lung and ovarian carcinomas.
The selection of many DNA reactive drugs for further in vivo testing was primarily based upon their high reactivity with DNA bases, their DNA binding affinity and on the significant cytotoxicity that they induce in tumor cells. Structure-activity relationship studies were often directed at cell growth and/or in vivo activity in murine models.
The over-expression and dysfunction of tyrosine kinases (TKs), directly or indirectly implicated in mitogenic signaling in tumor cells, have been extensively studied and are now considered the major functional differences between normal and tumor cells (1). Because of their significant involvement in tumor progression, over-expressed receptor TKs have now become the targets for drug design and selective chemotherapeutic interventions (2). One such target is the epidermal growth factor receptor (EGFR) which, in many patients, is associated with aggressive tumor progression and invasion (3). It has already been demonstrated that blocking signal transduction through the mediation of the TK activity of the EGFR translates into significant anti-tumor activity both in vitro and in vivo, and two novel agents are now in Phase II clinical trials (4). Despite being significantly less toxic than previous cytotoxic agents, most TK inhibitors currently in clinical trials have the disadvantage of being cytostatic agents that induce reversible inhibitory growth activity (5).
Many human tumors, including lung, breast and brain tumors, express high levels of the EGFR (6). It has already been shown that blockade of the EGFR pathway by several methods inhibits the proliferation of a variety of tumor cell lines both in vitro and in vivo (7-10). Although EGFR antibodies have recently been shown to trigger apoptosis in tumor cells (11), the anti-proliferative activity of EGFR tyrosine kinase inhibitors is often cytostatic and not cytotoxic.
The anti-tumor efficacy of EGFR TK inhibitors has already been demonstrated in vivo. However, one major limitation of current EGFR tyrosine kinase inhibitors is the high intracellular concentration of ATP which represents a major barrier to sustained inhibition of EGF-stimulated signal transduction in tumor cells. Where they cannot induce apoptosis, EGFR TK inhibitors are cytostatic agents that induce reversible antitumor effects. Considerable attention is now being turned towards the synthesis of irreversible inhibitors. Recently, irreversible inhibitors of the EGFR family have been synthesized showing greater potency than their reversible predecessors (12, 40, 41). The irreversible inhibition of TKs may nonetheless be insufficient to induce cell death. Unfortunately, irreversible inhibition of EGFR may not suffice to induce sustained antitumor activity if the cells possess alternative growth mechanisms. The combination with cytotoxic drugs, to potentiate the action EGFR TK inhibition, is now being considered a useful alternative (13).
Anilinoquinazolines are a novel class of highly specific receptor compounds that were shown to inhibit EGFR-related signal transduction by competitive binding in the ATP site of the EGFR (14). Blockade of EGFR-mediated signal transduction by anilinoquinazolines of types A and B has already been shown to translate into significant antitumor activity (7,8). The significant number of structure-activity-relationship (SAR) studies on 4-anilinoquinazolines and pyrido[d]pyrimidines as EGFR TK inhibitors is consistent with the compounds binding to the ATP site of the EGFR (7-10, 15).

Molecular modeling suggests that the N-1 atom accepts a H-bond from Met-769, whereas the N-3 atom accepts a H-bond from the side chain of Thr-766 located on strand 5 deep in the binding cleft (8,9,16). The anilino moiety binds in an adjacent hydrophobic pocket. Molecular modeling further suggests that the only positions on the inhibitors where substituents could be altered without affecting their binding affinity are the 6- and 7-positions located at the entrance of the binding cleft (17). A variety of compounds with bulky side chains on the 6- and 7-positions have been synthesized and were found to retain significant binding affinity for the EGFR ATP binding site. The tolerance of bulky substituents in the SAR of anilinoquinazolines was illustrated by the significant activities of structures A and B, which are now in Phase II clinical trials.
Nitrosoureas remain among the oldest drugs used in the clinical management of leukemias and many solid tumors (18-20). The lead compound of this class, bis-chloroethyl-N-nitrosourea (BCNU) has been one of the most potent agents used in the treatment of brain tumors (19,20) for over 30 years. Its mechanism of action is based on the induction of cytotoxic DNA single-strand breaks and DNA cross-links that ultimately lead to cancer cell death (21,22).

Alkyldiazonium compounds and precursors such as dacarbazine and TEM (Scheme 1) are cytotoxic agents whose mechanism of action is primarily based on the alkylation of DNA at the 6- or 7-position of guanine (23). TEM is now approved for the treatment of melanoma and brain tumors.

There exist many biochemical differences between normal cells and tumor cells. Our knowledge of these differences has significantly increased. One such difference is the over-expression and mutation of several signal transduction proteins. The dysfunction of the EGFR and related receptors occurs in many tumors, including lung, breast and ovarian cancers. Compounds with multiple intracellular targets would be expected to be more effective against resistant tumors. More specifically, the development of chemical agents that demonstrate the potential to simultaneously target one such protein, here the EGFR, as well as genomic DNA would be greatly desired. Compounds with multiple intracellular targets would fight cancer more aggressively and would consequently be more effective. This mixed targeting strategy is termed the “Combi-Targeting Concept” and its development provides an alternative to classical therapies involving the non-selective cisplatin and many other alkylating agents. In other words, combining such non-selective DNA damaging agents with an oncogenic tyrosine kinase targeting molecule would result in new molecules being more selective and effective towards tumors expressing these oncogenes.
There thus remains a need for cytotoxic agents that can synergize with ligands that inhibit cell proliferation or oncogenesis. More specifically, there remains a need for chemical agents that are capable of simultaneously targeting a specific molecule, such as the epidermal growth factor receptor (EGFR), and genomic DNA.
The present invention seeks to meet these and other needs.